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Ribonuclease A

Figure 1. Ribbon diagram of the three-dimensional structure of ribonuclease A.72 The inscriptions refer to the location of the eight cysteine residues that give rise to the four disulfide bonds, the two proline residues with cis peptide bonds, and the three residues most important for catalysis: His12, His119, and Lys41.

copy has also been used to characterize the structure of RNase B.6,69,70 Altogether, over 70 sets of three- dimensional coordinates related to RNase A have been deposited in the Brookhaven Protein Data Bank (www.pdb.bnl.gov).

RNase A is small. The mature enzyme, as secreted by exocrine cells of the bovine pancreas, has only 124 amino acid residues. RNase A contains 19 of the 20 natural amino acids, lacking only tryptophan. The molecular formula of the native, uncharged enzyme is C575H907N171O192S12. This formula corresponds to a molecular mass of 13 686 Da. As a small protein, RNase A became a target of synthetic chemists and was the first protein to succumb to total synthesis. This preparation had low, but measurable, ribonucle- olytic activity. 11,71

The overall shape of the enzyme resembles that of a kidney, with the active-site residues lying in the cleft (Figure 172). The predominant elements of secondary structure are a long four-stranded anti- parallel -sheet and three short R-helixes. The enzyme is cross-linked by four disulfide bonds, which involve all eight of its cysteine residues. The peptide bonds preceding two of the four proline residues are in the cis (or E) conformation. These proline residues are in type VI73 reverse turns at opposite ends of the native enzyme.

An important contribution to the understanding of RNase A function has been the determination of the structure of crystalline complexes between the en- zyme and nucleic acids that act as substrate or product analogues. Structures with oligonucleotides include those of RNase A with bound d(pA)4, d(pT)4,76 and d(ApTpApApG),77 and RNase B with bound d(pA)4.78 Structures with dinucleotides in- clude a productive (that is, catalytically meaningful) complex with d(CpA),79 and unproductive complexes with d(CpG) and cytidylyl(2f5)guanosine.80,81 Struc- tures of RNase A and its complexes, as revealed by X-ray diffraction analysis82 as well as NMR spectros- copy,83 have been the subject of recent reviews. 74,75

IV. Folding and Stability

The stability of RNase A is legendary. The clas- sical procedure for the purification of RNase A from

Chemical Reviews, 1998, Vol. 98, No. 3 1047

a bovine pancreas relies on the enzyme maintaining its integrity and solubility under drastic conditions: first, 0.25 N sulfuric acid at 5 °C, and then, pH 3.0 at 95-100 °C.84 The final step in this protocol calls for crystallization of the enzyme.

The three-dimensional structure of RNase A is fully encoded by its amino acid sequence.85-89 This dis- covery made RNase A into a favorite model system for the application of new methods to probe protein folding. In recent examples, electrospray mass spec- trometry has been used to determine which disulfide bonds (both native and nonnative) form during the folding of the reduced molecule90-92 or a derivative in which the eight cysteine residues are in mixed

disulfides with infrared (FTIR)

glutathione.92 Fourier transform spectroscopy, with its unique signa-

ture

for

  • -

    sheets,

has

been

used

to

probe

new

aspects

of RNase A folding.32,93-95

In these and other studies

on the folding of RNase A, the unfolded enzyme is generated by high or low temperature, high or low pH, or chaotropic agents. The unfolding of RNase A by high pressure has attracted much interest, prom- ising still more insights. 96-101

Two distinct starting materials have been used in most studies on the folding of RNase A: reduced enzyme and oxidized enzyme (with the four native disulfide bonds intact). Studies of the folding of the reduced enzyme have focused on disulfide bond formation. Studies of the folding of the oxidized enzyme have focused on prolyl peptide bond isomer- ization. These and other aspects of the folding of RNase A have been the subject of a recent review. 102

A. Disulfide Bond Formation

The four disulfide bonds in RNase A are critical to the stability of the native enzyme. Replacing any cystine with a pair of alanine103 or serine34,104 residues reduces the thermal stability of the enzyme. The two disulfide bonds (Cys26-Cys84 and Cys58-Cys110) between an R-helix and a -sheet contribute more to thermal stability than do the two disulfide bonds between (Cys40-Cys95) or within (Cys65-Cys72) a surface loop. 103

Disulfide bonds, as covalent but sometimes transi- tory cross-links,105 can be useful probes for elaborat- ing protein folding pathways. With RNase A as with other proteins, folding has been studied by allowing the reduced protein to be oxidized by small-molecule disulfides such as oxidized glutathione (or oxidized dithiothreitol106), quenching the incomplete reaction by acidification or alkylation, and identifying the disulfide bonds in the folding intermediates. Both the acquisition and interpretation of such data on RNase A have been controversial. (For a review, see ref 102.) The controversy is due to the complexity of forming the four native disulfide bonds from eight cysteine residues. This complexity arises because eight cysteine residues can form 28 () 8C2) distinct disulfide bonds. Moreover, a protein with eight cysteine residues can form 105 () 8C8 × 7 × 5 × 3) distinct species containing four disulfide bonds and 7 6 4 ( ) 8 C 8 × 7 × 5 × 3 + 8 C 6 × 5 × 3 + 8 C 4 × 3 + 8 C 2 + 8 C 0 ) altogether. distinct Indeed, oxidized RNase and reduced species, A with intentionally

scrambled substrate

disulfide bonds has become a conventional for enzymes, such as protein disulfide

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